This invention relates to the assay of free analytes. For the purposes of this invention the unbound free analyte shall be taken as a substance present in a sample in which a proportion of the substance is unbound and a proportion is bound to a receptor. Generally a predominant proportion of the substance will be reversibly, albeit firmly bound to the receptor. Also, in many instances the receptor will have sites available for binding additional analyte even though, according to the Law of Mass Action, there will be a very small amount of analyte free in solution. The receptor will ordinarily be soluble, and in most cases is a protein.
The importance of free analytes is that the physiological action of many drugs and hormones correlates much more closely with the concentration of free drugs or hormones in circulation than with their total levels. Examples of such free analytes and their receptors are thyroid hormones with thyroxine binding proteins (TBP); aldosterone, progesterone and testosterone with sex hormone binding globulin; diphenylhydantoin with albumin; antihemophilic factor with its inhibitors; proteolytic enzymes with their inhibitors or activators; cortisol with cortisol binding protein; and vitamin B.sub.12 with intrinsic factor. Many others are known or will become known in the future. The most thoroughly investigated and the most important of these systems is the thyroid hormone-TBP binding system. Since most of the prior art relates to this system, free thyroid assays are largely the subject of the following discussion. However, it should be noted that the same problems and methods will be relevant to the determination of other free analytes.
Functional thyrometabolic status has been commonly evaluated by measurement of the total serum content of the thyroid hormone thyroxine (3,5,3',5'-L-tetraiodothyronine, T4). Greater than 99.95% of the circulating T4 serves as a metabolically inert reservoir transported in serum bound primarily to thyroxine binding globulin (TBG), and secondarily to thyroxine binding prealbumin (TBPA) and albumin, collectively termed thyroxine binding protein (TBP). A great deal of evidence exists that the remaining less than 0.1% of the free or unbound fraction of circulating T4 is physiologically active because it diffuses into tissue to exert its action, its concentration is an important determinant of the hormone removal rate, and anomalies in TBP do not normally affect its concentration. It is thought to be the precursor of triiodothyronine (T3), which is also present in serum in the free and protein-bound forms. Again, the free T3 level correlates best with functional thyroid status.
The absolute quantity of T4 in samples, regardless of its free or bound status, has been measured by total T4 tests. The usual procedure involves denaturing TBP with alcohol or adding a displacing agent such as salicylate to release a large proportion of the T4 into solution, followed by addition of radioisotope labelled T4 and a T4 receptor such as an ion exchange resin or insoluble antibody. Sample and labelled T4 are allowed to compete for limited sites on the receptor, then the receptor is separated from the soluble reactants and the amount of radioactivity bound to the receptor is determined. This radioactivity is inversely proportional to the amount of sample T4 originally present. However, since total T4 rises and falls with the TBP, particularly TBG, levels in the euthyroid individual, an abnormal thyroid function may be erroneously indicated by any factor which changes the concentration of the binding proteins. Androgens, estrogens, anabolic steroids, glucocorticoids, and normal pregnancy alter the TBG levels. Thus the total T4 assay alone has not been found to be a reliable indicator of the thyrometabolic status.
Alterations in TBG levels are usually compensated for by a shift in the total T4 levels, as the negative feedback mechanism of the hypothalamic-thyroid axis works to keep the free T4 concentration constant. The unbound or "free" portion of thyroxine in serum is therefore the most useful constant indicator of thyrometabolic status. The amount of unbound thyroxine is governed by the Law of Mass Action, and is largely a function of the total circulating T4 and the concentration of TBG.
The difficulties of correlating thyrometabolic status with total T4 led to the development of the thyroid hormone uptake assay. This test measures the relative amount of serum TBP binding capacity, i.e., that which has not already been occupied by uptake of thyroid hormone in vivo. Thyroid hormone uptake immunoassays are ordinarily conducted by adding labelled T3 to a serum sample until the T3 is absorbed by the unsaturated TBP binding sites, scavenging the unabsorbed T3 from the reaction mixture by contacting the mixture with an insoluble T3 receptor such as an ion exchange resin or antibody and measuring the scavenged labelled T3. T4 may also be used. A proportionately large number of unoccupied binding sites will be indicated by a commensurately low amount of labelled thyroid hormone bound to the insoluble receptor.
In lieu of a direct free T4 measurement the percentage thyroid hormone uptake has been multiplied times the results of a total T4 or protein bound iodine assay on the same sample to arrive at the Free Thyroxine Index (Clark et al., "Journal Clin. Endocrinol." 25:39-45 [1965]). The Free Thyroxine Index is based on the premise that the uptake result is inversely proportional to the unsaturated TBP in serum and that free T4 varies directly with total T4 and inversely with unestimated TBP levels. The Free Thyroxine Index suffers from various disadvantages. It is capable of limited sensitivity and precision, separate determinations are required, and the uptake test upon which the Index is based may give erroneous values when used on sick euthyroid patients, persons with hereditary TBG deficiency, persons with increased TBP concentrations, and in those patients undergoing therapy with drugs such as phenylbutazone, diphenylhydantoin, salicylate and prednisone.
A requirement for two separate determinations is also an integral part of the kinetic free T4 assay disclosed in U.S. Pat. No. 4,046,876. Here two series of tubes are assayed. In both series an equilibrium is first established between labelled T4, endogenous sample T4 and TBP. In one series an agent is present to displace a large proportion of the T4 from TBP, i.e., this series produces a determination of total T4. Since the other series contains no such agent it will essentially yield a T4 uptake determination. Then each reaction mixture is added to insoluble T4 antibody, incubated for a set period, the phases separated and the solid phase assayed for the label. The relative reaction rates in the two series are calculated for each pair of tubes and the results compared with a standard plot. Aside from the disadvantages of requiring two sets of assays for each determination, as with the closely related Free Thyroxine Index, this method requires tedious calculations. Also, since it is a kinetic method it is highly time dependent, thus making it difficult to accurately conduct large numbers of determinations simultaneously.
U.S. Pat. No. 3,799,740 discloses a technique for obtaining in a single container a measure of free T4, the Effective Thyroxine Ratio. However, the method is still essentially based upon a dual assessment of thyroid hormone uptake and total T4. In accordance with this method sample T4 is extracted from TBG and an aliquot of this extract is combined with a complex of labelled T4 with TBG and with an aliquot of unsaturated TBG from the same sample. An anion-selective resin is then added to the reaction mixture, incubated for a carefully controlled period, the resin removed and the amount of label in the supernatant determined. While a portion of the analytical steps of this method may be performed in a single container the T4 extraction procedure is burdensome and introduces considerable error into the determination. Other error is introduced during the handling of two sample aliquots. Finally, the method is overall procedurally complex.
Similarly, U.S. Pat. No. 3,941,504 discloses a free T4 assay which may be conducted within a single container. Here a sample and an aliquot of labelled T4 are combined, then the labelled and unlabelled T4 are extracted from the serum TBP on a dextran gel column at high pH. A percentage of the total added label is retained by the column upon elution with unsaturated TBP and a portion of the pre-extracted sample. This percentage, the Free Thyroxine Equivalent, is an indirect measure of free T4 which may be quantitated by comparison with appropriate standards. While this method does not expressly call for a total T4 assay as part of the procedure it nonetheless suffers from the same errors introduced during the extraction of T4 and labelled T4 from TBG on the dextran gel column. Further, column chromotography is inconvenient, imprecise and not suited to large scale clinical use. This method also entails handling duplicate patient samples, a ready source for error, and includes an undue number of manipulative steps.
Another group of related assays, generically termed the free thyroxine tests, also requires determination of the total endogenous T4. In these tests serum isincubated with labelled T4 until labelled and unlabelled T4 are uniformly distributed between TBP and the free proportion of T4. Simultaneously with or following this incubation the unbound T4 is separated from the TBP-bound fraction using a variety of techniques, e.g., charcoal or dextran gel absorption, ultrafiltration or dialysis. Again, the result is mathematically combined with the results of a total T4 determination on the same sample. These tests therefore suffer from the principal defect noted for other free T4 methods, i.e., the need for a total T4 assay to quantitate the percentage free T4 yielded by the other member of the dual assay. This multiplies the errors involved in each test and doubles the expense and time required. Some special problems are also encountered with these methods, e.g., the need for highly purified labelled T4 in the dialysis and ultrafiltration techniques. Examples of these methods are Oppenheimer et al, "Journal of Clinical Investigation" 42(11):1769-1782 (1963) and Cavalieri et al, "Journal of Nuclear Medicine" 10(9):566-570 (1969). A similar assay for free testosterone using dialysis separation is known.
Finally, free T4 has been directly determined by dialyzing a sample and assaying the dialysate by gas chromatography or electrophoresis. These methods lack the sensitivity, precision and simplicity of binding assays, require a great deal of technical skill and are too slow for routine laboratory use.